Interferon Inhibition in SLE: From Bench to Bedside

Despite advances in the management of systemic lupus erythematosus (SLE), it remains a chronic disease with frequent flares, requiring constant medical care, laboratory exams, hospitalisations, and the use of immunosuppressive drugs and corticosteroids, increasing the morbidity and mortality of these patients. The past decade of research has brought to light multiple observations on the role of interferons (IFNs) in the pathogenesis of SLE, which paved the way for the development of potential novel therapies targeting the interferon pathway. Following two phase III trials, anifrolumab, a monoclonal antibody which binds to the type I IFN receptor, blocking the activity of type I IFNs, was approved for active SLE. This review summarises the latest research on the role and mechanisms of type I IFNs in SLE and the development and advances on new therapeutic drugs based on IFN inhibition for SLE.


INTRODUCTION
Systemic lupus erythematosus (SLE) is a complex autoimmune disease, characterised by great clinical heterogeneity as it can affect literally every system and organ with varying degrees of severity.It predominantly affects women of reproductive age, with an estimated female to male ratio of 9:1, while ten to twenty percent of all patients with SLE are diagnosed during childhood, with a lower female/male ratio and approximately 10 percent have disease onset after the age of 50. 1,2espite advances in the diagnosis and management of SLE, it remains a chronic disease with a course characterised by alternating periods of remission and relapse and significant morbidity due to irreversible organ damage as a result of chronic inflammation, corticosteroid use and comorbidities. 3,4Therefore, the development of novel therapies that will achieve long periods of remission with reduced corticosteroid use, remain an unmet need.The approval of belimumab, the first biologic therapy for SLE, marked a major advance in the management of the disease, paving the way for the development of further biological agents.Belimumab is an anti-B lymphocyte stimulator monoclonal antibody 5 and was approved for the treatment of active SLE in 2011, further extended for treatment of active lupus nephritis (LN) as an add-on to standard-of-care (SOC). 6ore recently, literature has emerged that offers multiple observations on the role of interferons (IFNs), a type of cytokines important for fighting viral infections and regulating the immune system, in the pathogenesis of SLE. 7 Following two phase III trials, anifrolumab, a monoclonal antibody which binds to type I IFN receptor subunit 1 (IFNAR1), inhibiting the activity of type I IFNs, was approved for active SLE. 8n this review, the latest research about the role and mechanisms of type I IFNs in SLE development and advances on new therapeutic drugs based on IFN inhibition for SLE are summarised.

THE FAMILY OF INTERFERONS
Interferons belong to a family of signalling proteins released by host cells in response to the presence of pathogens, typically during viral and bacterial infections. 9heir name derives from their ability to "interfere" with virus replication by protecting cells from viral infections. 10owever, their function is not limited to fighting pathogens, since they also have antitumor, antiproliferative and immunomodulatory effects. 11More specifically, IFNs activate immune cells, such as natural killer (NK) cells and macrophages, upregulate antigen presentation by increasing major histocompatibility complex (MHC) antigen expression, thereby increasing host defence. 9ased on their distinct structures, biological activities and the type of receptor through which they signal, human interferons have been classified into three major types: type I (alpha, beta, epsilon, kappa and omega), type II (gamma) and type III (lambda). 9Type I IFN alpha (IFN-α) are further divided in 12 different subtypes. 12FNs belonging to all three classes are important both for fighting viral infections and for the regulation of the immune system.Virtually all cell types can express type I and III IFNs, following recognition of viral components, especially nucleic acids, by cytoplasmic and endosomal receptors, although the plasmacytoid dendritic cell (pDC) is considered the natural IFN-producing cell.Emerging evidence shows that cellular sources of IFNs can vary during different viral infections. 13Specifically, during respiratory infections, cells lining the airways, like epithelial cells and alveolar macrophages, provide the primary source of type I IFN, during rotavirus infections, epithelial cells of the gastrointestinal system produce type I IFN, while neurons are critical sources of IFN-I during specific brain infections. 13In SLE, there is emerging evidence that apart from pDCs, other types of cells, such as keratinocytes, monocytes and neutrophils are involved in IFN production. 14On the other hand, type II interferon expression is restricted to immune cells such as T cells and NK cells and is induced by cytokines such as IL-12. 13,15N SIGNALLING Induction of type I IFNs typically occurs when pattern recognition receptors (PRRs) localised in the cytoplasm or in the endosome of cells, sense the presence of viruses, bacteria or microbial nucleic acids.14,16 These PRRs include membrane bound Toll-like receptors (TLRs), the cytoplasmic receptors retinoic acid inducible gene 1 (RIG-I)-like receptors (RLRs) and nucleotide oligomerisation domain-like receptors (NLRs).14,16 After their release, Type I IFNs all bind to the same ubiquitously expressed type I IFN receptor (IFNAR) that consists of two polypeptide chains of IFNAR1 and IFNAR2.Subsequently, the signalling pathway involves activation of Janus kinase (JAK) 1 and tyrosine kinase (TYK) 2 and formation of the interferon-stimulated gene factor 3-complex (IGSF3), which includes signal transducer and activator of transcription (STAT)1, STAT2 and interferon regulatory factor (IRF).IGSF3 moves into the cell nucleus and binds to specific nucleotide sequences, called IFN-stimulated response elements (ISREs), which induce new gene transcription (IFN-regulated genes) in order to mediate antiviral responses.12 The type II IFN comprise of IFN-γ which binds to the IFN-γ receptor (IFNGR) which is expressed on most cells.15,17 Ligation of the IFNGR results in phosphorylation of STAT1 homodimers, via activation of JAK1 and JAK2, and binding to IFN-γ-activated sites (GASs) and subsequent gene expression.Induction of type I and II genes is largely overlapped, since the latter's signalling pathway can also be used by IFNAR.15 Type III IFNs consist of four newly identified lambda IFNs: IFNλ1/IL29, IFNλ2/IL28A, IFNλ3/IL28B and IFNλ4.16,18 IFN-λs are mostly found at barrier surfaces and are produced by epithelial and epithelial-origin cells of the respiratory and gastrointestinal tracts.The type III IFNs signal through a receptor complex (IFNLR1/IL10Rβ) that is primarily expressed on gastrointestinal, respiratory and urogenital epithelial cells, hepatocytes and a few immune cells including neutrophils and DCs.16,18 IFN SIGNATURE IFNs have been intensively investigated recently due to their crucial role in a number of immunological pathways involved in autoimmune diseases, summarised by the term "IFN signature". The s-called IFN signature refers to the evidence of an upregulation of transcripts induced by the different IFN subtypes.17,19 Until recently, the term did not allow differentiation between the three families of IFNs, whereas in recent literature both the terms "IFN signature" and "type I IFN signature" are used to encompass the overexpression of genes induced by type I IFNs.18,20 Diseases in which this signature appears to play a prominent role are SLE, Sjögren's syndrome, inflammatory myositis and scleroderma.17,19,[21][22][23][24] Although extensive research has been conducted, there is still debate about various aspects regarding the measurement of the IFN signature. Firstof all, there is no universally accepted combination of genes to be analysed for the calculation of the IFN score, which is classically assessed by the expression level of different IFN-induced mRNA.17,19 In the most recent literature, four or five gene sets 19,21 have been used when evaluating the IFN signature in autoimmune rheumatic diseases (ARDs).In addition, the transcriptomic overlap between distinct types of IFNs, is still a limitation, 23,25 although the development of new techniques allows the differentiation of type I IFNs.24,26 The type of IFN producer cells to analyse has also shifted from the initial focus on plasmacytoid DCs to tissue-resident immune cells, keratinocytes, renal tubular cells, salivary gland epithelial cells and neutrophils.[25][26][27][28][29] The causes or triggers of the IFN signature in ARDs are also not clear.12 Finally, the implications of IFN signature measurement in clinical practice needs to be explored, in order to determine its relevance for patient stratification and optimisation of ARD management.17,19 IFNs IN SLE SLE patients are characterised by increased levels of IFN in serum, a fact that has been known since the late 70s.28,30 Inherited mutations causing activation of the type I IFN pathway result in a lupus-like phenotypic activation of systemic autoimmunity.29,31 Expression level of IFNinduced genes correlates with SLE activity and severity, including active renal disease.12 As previously mentioned, although pDCs are probably the main source of IFN production in SLE, several other cell types contribute to the IFN signature, either by producing IFN themselves, or by stimulating pDC to an increased IFN production, contributing to the sustained autoimmune process in SLE. 12 Circulating immune-complexes (ICs) seem to play a major role in the excessive activation of pDCs in SLE.This is supported by in vitro studies showing that DNAcontaining ICs from active SLE patients' serum, activate the innate immune system by inducing pDCs to produce IFN-α, and other pro-inflammatory cytokines and chemokines.30,32 Studies on healthy first-degree relatives of SLE patients have shown elevated serum IFN-α levels compared to healthy unrelated individuals .31,33suggesting that an underlying genetic susceptibility is also required for producing high IFN-α levels in SLE.

IFNs have pleiotropic actions on various innate and adaptive immunity cells, namely activation and differentiation of B cells into plasma cells, increased T-cell proliferation and activation, impaired function of regulatory
T-cells, and BAFF upregulation by dendritic cells, all of which contribute to SLE pathogenesis. 32,34This high IFN signature seems to have a major impact on the full range of clinical manifestations in SLE.Specifically, increased expression of IFN-regulated genes has been observed in epidermis and dermis of cutaneous lesions 33,35 and the IFN signature has been demonstrated to correlate with cutaneous disease activity, suggesting a key role of IFN signalling in SLE skin pathology. 34,36However, the exact interplay between different IFNs, keratinocytes and pDCs needs further exploration.Increased expression of IFNinduced genes has been also demonstrated in synovial tissue from patients with SLE and inflammatory arthritis, probably deriving from fibroblasts, which are abundant in this tissue. 35,37he IFN-signature seems to also contribute to the severe SLE manifestations.Specifically, kidney biopsies of patients with lupus nephritis have shown increased expression of IFN-inducible genes, while pDCs accumulate in glomeruli of patients with active renal disease. 36,38oreover, high IFN expression in peripheral blood correlates with LN severity. 37,390][41] In addition, IFN-α has been shown to activate microglia leading to synaptic pruning in lupus-prone mouse models and therapeutic administration of type I interferons induces psychiatric symptoms. 38,40

TARGETING THE IFN SYSTEM
It is now understood that IFNs play a critical role in the pathogenesis of SLE, which explains the numerous attempts to develop agents that inhibit the IFN pathway during the past decades.[42][43] Anifrolumab Anifrolumab, previously known as MEDI-546, is the first biologic targeting the IFN system to be approved for SLE. 42,44It is a fully human, IgG1κ monoclonal antibody, able to bind to IFN-α/β receptor (IFNAR), leading to prevention of signal transmission by all type I IFNs. 42,44ecent phase II and III trials have proved its efficacy and safety in active SLE, [41][42][43][44][45] summarised in Table 1.MUSE was a phase IIb, randomised, double-blind, placebo-controlled study, 42,44 which evaluated anifrolumab's efficacy and safety in adult SLE patients with moderate to severe disease activity, as an add-on to SOC.More patients on anifrolumab achieved a SLE Responder Index (SRI) 4 response 44,46 at week 24 compared to placebo.Serious adverse event rates were similar across groups; however, herpes zoster and influenza incidence were more frequent on anifrolumab treated patients.A post hoc analysis of the MUSE study 45,47 showed a greater response in rash and arthritis resolution in anifrolumab treated patients with high IFN signature, while an open label extension study showed sustained disease activity with comparable serious adverse events to those reported in the randomised controlled trial (RCT) phase. 46,48ULIP-1 was the first phase III RCT to be conducted on the use of anifrolumab in active SLE, whose primary endpoint was not met. 47,49However, several secondary endpoints were reached, which led to TULIP-2, the second phase III RCT on anifrolumab, with similar design, 43,45 where a different primary endpoint, than in the TULIP-1 was used, the British Isles Lupus Assessment Group-Based Composite Lupus Assessment (BICLA) response. 48,50In this study, patients received placebo (n=182) or 300mg anifrolumab (n=180) every 4 weeks for a year's period.The percentage of patients with a BICLA response was greater in the anifrolumab than in the placebo group (47.8% vs 31.5% respectively).Some secondary end points were also reached, including steroid dose reduction and Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) 49,51 improvement.Herpes zoster, upper respiratory tract infection and bronchitis were more frequent in the anifrolumab arm.Post-hoc analysis of both TULIP trials showed diminished overall disease activity combined with sustained glucocorticoid tapering in patients on anifrolumab compared to placebo 50,52 and lower annualised flare rates, with longer periods of remission. 51,53A long-term extension of both TULIP trials (TULIP LTE) further assessed its safety for an additional 3 year period, showing no alterations in the safety profile of anifrolumab, with SAE, malignancy and major cardiovascular events being equivalent across the two arms. 52,54ollowing approval of anifrolumab for SLE patients with non-renal, active SLE, the first attempt to assess its efficacy in patients with active, biopsy proven, Class III/IV LN in the TULIP-LN phase II RCT 53,55 did not achieve the desired results.Patients were assigned to receive either placebo or anifrolumab added to SOC treatment with the change in baseline 24-hour urine protein-creatinine ratio (UPCR) at week 52 as the primary endpoint.][55][56] Finally, a phase II pharmacokinetics/pharmacodynamics (PK/PD), safety and efficacy, RCT evaluated the subcutaneous administration of anifrolumab in SLE patients with active skin disease and high Type I IFN levels, over SOC treatment. 55,57Overall, PK/PD, safety and efficacy profile, supported the further development of subcutaneous anifrolumab for SLE treatment.Several ongoing trials on anifrolumab, summarised in Table 2, are expected to further characterise its profile.Importantly, anifrolumab on active proliferative nephritis will be evaluated in a phase III trial (IRIS, NCT05138133). 56,58n open label study from Japan will assess anifrolumab administration early in disease course, prior to other immunosuppressive or immunomodulatory drugs (jRCTs031230358), 57,59 while further studies on subcutaneous anifrolumab administration will be conducted on moderate to severe SLE (TULIP SC NCT04877691), 58,60 as well as on cutaneous lupus erythematosus refractory to first treatment line (LAVENDER, NCT06015737). 59,61inally, a phase III PK/PD, efficacy and safety trial on anifrolumab administration in paediatric SLE is expected to start recruiting soon (NCT05835310). 60,62ti-IFN monoclonal antibodies Two monoclonal antibodies targeting specifically IFN-α, sifalimumab and rontalizumab, have been studied in phase I and II clinical trials.Sifalimumab is a human monoclonal antibody that directly targets IFN-α. 61,63Several RCTs [61][62][63][64][65] and an open label study 64,66 showed promising results regarding tolerability and safety of sifalimumab, while its primary endpoint was met in a phase 2 study, with a higher percentage of patients achieving SRI-4 in the treatment group. 63,65However, its development was discontinued in favour of anifrolumab, which showed more favourable results as described above. 41,43,63,65ontalizumab, a humanised IgG1 monoclonal antibody, designed to neutralise all known IFN-α subtypes, 65,67  and a phase II study in SLE patients, [65][66][67][68] while its efficacy was not proved as compared to placebo, leading to discontinuation of its development.
Another human monoclonal antibody targeting the majority of IFN-α subtypes, as well as IFN-ω, JNJ-55920839, 67,69 was well tolerated in healthy adults and SLE patients with mild to moderate disease activity in phase I study, [67][68][69][70] while an improvement in several disease indexes was recorded. 68,70There are no registered phase II studies for this agent yet.Finally, although AGS-009, a humanised anti-INFα monoclonal antibody neutralising various IFN-α subtypes, showed good safety profile in a phase Ia RCT in adults with mild to moderate SLE, 40,42 it was not further developed.

IFNα-kinoid
IFNα-kinoid (IFN-K) is a vaccine constructed as a therapeutic agent combining inactivated IFN-a2b with a T-helper carrier protein.Mathian et al. uncovered its potency to induce polyclonal antibodies neutralising all 13 subtypes of human IFN-α in human IFN-α transgenic mice, without affecting IFN-β or IFN-γ. 69,71Results from a multicentre, phase I/IIa staggered dose-escalation trial in adult SLE patients immunised with IFN-K, proved its efficacy on developing anti-IFNα antibodies.IFN signa-ture-positive patients had both higher anti-IFNα titres and a reduced expression of IFN-induced genes.Higher anti-IFNα antibody titre was associated with IFN score decrease and C3 complement increase. 70,72An extension of this study further showed a diminished expression of genes involved in B cell activation following IFN-α neutralisation, and that antibody response induced by IFN-K had a polyclonal effect on 13 IFNα subtypes. 71,73Severity of adverse events in terms of injection site or systemic reactions was mild or moderate. 70,72nother phase IIb RCT, showed a reduction of IFN gene signature and a strong polyclonal immunogenic response in 91% of immunised patients.Despite the fail to meet a favourable change in response rate measured by BICLA, Lupus low disease activity state (LLDAS) was achieved in more patients administered IFN-K than placebo, also allowing more steroid reduction. 72,74Recorded adverse events were about the same among the two arms of the study, namely mild infections, headaches, nasopharyngitis and arthralgia and injection site induration. 72,74direct targets of the IFN system Several other agents indirectly affect the IFN pathway, among which JAK/TYK inhibitors, immunomodulators that are successfully used for the treatment of several

TITLE
autoimmune diseases, such as rheumatoid arthritis. 75By inhibiting the activity of one or more of the JAK family of enzymes (JAK1, JAK2, JAK3, TYK2), they interfere with the JAK-STAT signalling pathway in lymphocytes and downregulate IFN signalling. 75In SLE patients, Deucravacitinib, a TYK 2 inhibitor and tofacitinib both managed to reduce type I interferon gene signature in early studies. 76,77Contrarily, several other trials testing JAK inhibitors in SLE patients failed to reach the prespecified endpoints. 78,79itifilimab (BIIB059) is a humanised IgG1 monoclonal antibody targeting blood dendritic cell antigen 2 (BDCA2) reducing, among other cytokines, plasmatocytoid dendritic cells' type I IFN production, which has already proved efficacious in a phase II trial of CLE patients. 80][83][84] Dapirolizumab pegol (DZP) is a polyethylene glycol-conjugated antigen-binding fragment, targeting CD40L which, after its proved efficacy in active SLE, 85 showed decreased expression of type I IFN signature in patients with high baseline type I IFN expression. 86Finally, GSK2646264, a spleen tyrosine kinase (SYK) inhibitor with topical application, showed a modest incline of several interferon-related genes, in patients with cutaneous lupus erythematosus (NCT02927457). 87

CONCLUSIONS
Our understanding of how the IFN system impacts on the sustained autoimmune process in SLE continues to progress.Nevertheless, there are still challenges to overcome to unlock the complexity of targeting the IFN pathway in a multifaceted disease like SLE.Further clinical trials on the use of anifrolumab in severe SLE complications and results from ongoing trials of novel IFN inhibitors are eagerly awaited.

AUTHOR CONTRIBUTIONS
DD and CA acquisition, analysis and interpretation of data, manuscript drafting and critical revision for important intellectual content.ES analysis and interpretation of data, manuscript drafting and critical revision for important intellectual content; All the authors have read and approved the final version of the manuscript and agreed to take full responsibility for the integrity and accuracy of all aspects of the work.

Figure 1 .
Figure 1.Induction mechanism of genes by type I interferons.After their release, Type I IFNs all bind to the same ubiquitously expressed type I IFN receptor (IFNAR) that consists of two polypeptide chains of IFNAR1 and IFNAR2.Subsequently, the signalling pathway involves activation of Janus kinase (JAK) 1 and tyrosine kinase (TYK) 2 and formation of the interferon-stimulated gene factor 3-complex (IGSF3), which includes signal transducer and activator of transcription (STAT)1, STAT2 and interferon regulatory factor (IRF).IGSF3 moves into the cell nucleus and binds to specific nucleotide sequences, called IFN-stimulated response elements (ISREs), which induce new gene transcription (IFN-regulated genes) in order to mediate antiviral responses.

Table 1 .
demonstrated an acceptable safety profile in a phase I Completed trials on anifrolumab.
Yes CLASI; Cutaneous Lupus erythematosus Disease Area and Severity Index, LN; Lupus Nephritis, LTE; Long Term Extension Study, NA; Not Applicable, PK; Pharmacokinetics, SLE; Systemic Lupus Erythematosus TITLE